The control of cell growth and differentiation requires specific factors which exert their effects via interaction with receptors on the surface of responsive cells. Despite the increasing number of growth and differentiation factors that have been discovered and characterized, the precise structures involved in binding and biological activity and the sequential and causal molecular events underlying the activation of multiple receptors are largely unknown.
Nerve growth factor (NGF)is a 118 amino acid polypeptide which controls the survival, development and differentiation of the sympathetic nervous system, as well as parts of the sensory and central nervous systems (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980; Whittemore and Seiger, 1987; Thoenen et al., 1987). The biologically active form of NGF is a dimer of identical subunits each of which is produced from a precursor molecule (Angeletti and Bradshaw, 1971; Angeletti et al., 1973). A cDNA clone for NGF was first isolated in the mouse (Scott et al., 1983). Subsequently, the NGF gene has been characterized in a number of other species including several mammals, birds, reptiles and fishes (Schwarz et al., 1989; Hallbook et al., 1991).
NGF belongs to a family of structurally and functionally related molecules, collectively known as neurotrophins of the nerve growth factor family, which includes at least three other members, brain-derived neurotrophic factor (BDNF) (Barde et al., 1982; Leibrock et al., 1989), neurotrophin-3 (NT-3) (Hohn et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990; Ernfors et al., 1990) and neurotrophin-4 (NT-4) (Hallbook et al., 1991; Ip et al., 1992).
NGF interacts with a low-affinity receptor expressed on a variety of cell types of both neuronal and non-neuronal origin (Ernfors et al., 1988; Yan and Johnson, 1988; Heuer et al., 1990; Hallbook et al., 1990). The other three neurotrophins of the nerve growth factor family can also bind to the low-affinity NGF receptor (Rodriguez-Tebar et al., 1990; Ernfors et al., 1990; Squinto et al., 1991; Hallbook et al., 1991). This receptor is represented by a transmembrane glycoprotein of approximately 75,000 daltons (p75.sup.NGFR) which binds NGF with a Kd of 10-9M (Johnson et al., 1986; Radeke et al., 1987). However, high affinity binding (Kd=10-.sup.11 M), restricted to a subpopulation of p75.sup.NGFR -positive cells, is necessary to mediate the biological action of NGF. Banerjee et al., 1973; Herrup and Shooter, 1973; Sutter et al., 1979; Richardson et al., 1986). While the molecular relationship between the two receptor states is not entirely clear, several reports have indicated that the cytoplasmic domain of p75.sup.NGFR which lacks structural features known to mediate signal transduction in other receptors, is required for high-affinity binding and signal transduction (Hempstead et al., 1989; Yan et al., 1991; Berg et al., 1991).
It has recently been demonstrated that the proto-oncogene trk encodes a functional receptor for NGF (Kaplan et al., 1991a; Klein et al., 1991). The product of the trk proto-oncogene is a 140,000 dalton protein (p140.sup.trk) which is a member of the tyrosine kinase family of transmembrane receptors (Martin-Zanca et al., 1991). Though it has been postulated that this protein participates in the primary signal transduction mechanism of NGF, there is considerable disagreement regarding the equilibrium binding constant of p140.sup.trk for NGF. Whereas Klein et al. (1991) reported that p140.sup.trk binds NGF with both low and high affinities, Kaplan et al (1991) and Hempstead et al (1991) reported that p140.sup.trk binds NGF with an affinity similar to that of p75.sup.NGFR and that coexpression for both receptors is required for high affinity binding to occur. Recently, the product of the trk proto-oncogene has been shown to constitute a functional receptor for NGF (Kaplan et al., 1991a; Klein et al., 1991). NGF binding to p140.sup.trk results in rapid phosphorylation of this molecule and stimulation of its tyrosine kinase activity (Kaplan et al., 1991a; Kaplan et al., 1991b; Klein et al., 1991).
In contrast, the role of p75.sup.NGFR in signal transduction has remained elusive. Recently, it was reported that the cytoplasmic domain of this receptor is involved in mediating neuronal differentiation (Yan et al., 1991) and NGF induced tyrosine phosphorylation (Berg et al., 1991) in PC12 cells. However, other recent studies have shown that polyclonal antibodies against p75.sup.NGFR abolish NGF binding to this molecule and some of the high-affinity binding but do not inhibit biological responses to NGF (Weskamp and Reichardt, 1991). Recent reports using cell lines expressing p140.sup.trk have demonstrated that in the presence of NGF this receptor molecule can mediate survival and mitotic proliferation of fibroblasts in the absence of p75.sup.NGFR (Cordon-Cardo et al., 1991). These studies could not rule out the possibility that binding to p75.sup.NGFR could be important in mediating NGF responses in neurons and neuron-like cell lines. It has also recently been shown that the trk proto-oncogene can rescue NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines (Loeb et al., 1991). However, these cells still expressed substantial levels of p75.sup.NGFR therefore making it difficult to assess whether the presence of this molecule was required for the observed functional effects.
A better understanding of the molecular mechanism by which NGF exerts its biological effects is provided by the study of structure-function relationships and the creation of NGF mutants with altered properties. Initial studies along this line have analyzed the functional importance of highly conserved amino acid residues in the chicken NGF (Ibanez et al, 1990). More recently, an analysis of chimeric molecules between NGF and BDNF has delineated regions involved in determining the biological specificities of these two factors (Ibanez et al 1991a). Comparison of NGF genes from different species has revealed clusters of amino acid residues which are highly conserved across different groups of vertebrates (see FIG. 1, which demonstrates the conservation of amino acid residues 25 to 36 (single letter code) in NGFs from different species and in the homologous region of different neurotrophins. FIG. 1A shows alignment of residues 25 to 36 from rat (SEQ ID NO: 1) (Whittemore et al., 1988), mouse (SEQ ID NO: 1) (Scott et al., 1983), human (SEQ ID NO: 1) (Ullrich et al., 1983), bovine (SEQ ID NO: 1) (Meier et al., 1986), guinea pig (SEQ ID NO: 1) (Schwarz et al., 1989), chicken (SEQ ID NO: 1) (Ebendal et al., 1986; Meier et al., 1986), xenopus (SEQ ID NO: 2) (ref) and snake (SEQ ID NO: 3) (Selby et al., 1987) NGF. FIG. 1B shows alignment of residues 25 to 36 from rat NGF (SEQ ID NO: 1) and the homologous residues of rat BDNF (SEQ ID NO: 4) (Maisonpierre et al., 1990), rat NT-3 (SEQ ID NO: 5) (Maisonpierre et al., 1990; Ernfors et al., 1990) and xenopus NT-4 (SEQ ID NO: 6) (Hallbook et al., 1991).
Among these conserved parts, the region panning residues 25 to 36 is the most hydrophilic and therefore likely to be on the surface of the NGF molecule (Meier et al., 1986; Ebendal et al., 1989). Synthetic peptides designed from this sequence have been shown to inhibit the in vitro biological activity of NGF (Longo et al., 1990).